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'udies on clubroot of 
Cruciferous plants 



A THESIS 

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL 
OF CORNELL UNIVERSITY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



BY 



CHARLES CHUPP 



Published as N. Y. (Cornell) Agr. Exp. Sta. Bui. 387, 1917. 



STUDIES ON CLUBROOT OF 
CRUCIFEROUS PLANTS 



A THE5I5 

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL 
OF CORNELL UNIVERSITY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



BY 

CHARLES CHUPP 



Published as N. Y. (Cornell) Agr. Exp. Sta. Bui. 387, 1917- 






3>^<,^.W*<^.,>^ 






MARCH, 1917 BULLETIN 387 

CORNELL UNIVERSITY 
AGRICULTURAL EXPERIMENT STATION 



STUDIES ON CLUBROOT OF CRUCIFEROUS 

PLANTS 



CHARLES CHUPP 



ITHACA, NEW YORK 
PUBLISHED BY THE UNIVERSITY 



CORNELL UNIVERSITY 
AGRICULTURAL EXPERIMENT STATION 

Experimenting Staff 

ALBERT R. MANN, B.S.A., A.M., Acting Director. 

HENRY H. WING, M.S. in Agr., Animal Husbandry. 

T. LYTTLETON LYON, Ph.D., Soil Technology. 

JOHN L. STONE, B.Agr., Farm Practice. 

JAMES E. RICE, B.S.A., Poultry Husbandry. 

GEORGE W. CAVANAUGH, B.S., Agricultural Chemistry. 

HERBERT H. WHETZEL, M.A., Plant Pathology. 

ELMER O. PIPPIN, B.S.A., Soil Technology. 

G. F. WARREN, Ph.D., Farm Management. 

WILLIAM A. STOCKING, Jr., M.S.A., Dairy Industry. 

WILFORD M. WILSON, M.D., Meteorolog>'. 

RALPH S. HOSMER, B.A.S., M.F., Forestry. 

JAMES G. NEEDHAM, Ph.D., Entomology and Limnology. 

ROLLINS A. EMERSON, D.Sc, Plant Breeding. 

HARRY H. LOVE, Ph.D., Plant Breeding. 

ARTHUR W. GILBERT, Ph.D., Plant Breeding. 

DONALD REDDICK, Ph.D., Plant Pathology. 

EDWARD G. MONTGOMERY, M.A., Farm Crops. 

WILLIAM A. RILEY, Ph.D., Entomology. 

MERRITT W. HARPER, M.S., Animal Husbandry. 

JAMES A. BIZZELL, Ph.D., Soil Technology. 

GLENN W. HERRICK, B.S.A., Economic Entomology. 

HOWARD W. RILEY, M.E., Farm Mechanics. 

CYRUS R. CROSBY, A.B., Entomology. 

HAROLD E. ROSS, M.S.A., Dairy Industry. 

KARL McK. WIEGAND, Ph.D., Botany. 

EDWARD A. WHITE, B.S., Floriculture. 

WILLIAM H. CHANDLER, Ph.D., Pomology. 

ELMER S. SAVAGE, M.S.A., Ph.D., Animal Husbandry. 

LEWIS KNUDSON, Ph.D., Plant Physiology. 

KENNETH C. LIVERMORE, Ph.D., Farm Management. 

ALVIN C. BEAL, Ph.D., Floriculture. 

MORTIER F. BARRUS, Ph.D., Plant Pathology. 

CLYDE H. MYERS, M.S., Ph.D., Plant Breeding. 

GEORGE W. TAILBY, Jr., B.S.A., Superintendent of Livestock. 

EDWARD S. GUTHRIE, M.S. in Agr., Ph.D., Dairy Industry. 

JAMES C. BRADLEY, Ph.D., Entomology. 

PAUL WORK, B.S., A.B., Vegetable Gardening. 

JOHN BENTLEY, Jr., B.S., M.F., Forestry. 

EARL W. BENJAMIN, Ph.D., Poultry Husbandry. 

EMMONS W. LELAND, B.S.A., Soil Technology. 

CHARLES T. GREGORY, Ph.D., Plant Pathology. 

WALTER W. FISK, M.S. in Agr., Dairy Industry. 

ARTHUR L. THOMPSON, Ph.D., Farm Management. 

ROBERT MATHESON, Ph.D., Entomology. 

MORTIMER D. LEONARD, B.S., Entomology. 

FRANK E. RICE, Ph.D., Agricultural Chemistry. 

VERN B. STEWART, Ph.D., Plant Pathology. 

IVAN C. JAGGER, M.S. in Agr., Plant Patholog>' (In cooperation with Rochester University). 

WILLIAM I. MYERS, B.S., Farm Management. 

LEW E. HARVEY, B.S., Farm Management. 

LEONARD A. MAYNARD, A.B., Ph.D., Animal Husbandry. 

LOUIS M. MASSEY, A.B.-, Ph.D., Plant Pathology. 

BRISTOW ADAMS, B.A., Editor. 

LELA G. GROSS, Assistant Editor. 

The regular bulletins of the Station are sent free on request to residents of New York State. 



CONTENTS 

PAGE 

Dissemination 421 

vSpore germination 42,^ 

Penetration 427 

Distribution within the host tissues . 434 

Spore formation and size 441 

A similar organism 442 

Bacteria in relation to Plasmodiophora Brassicae 443 

Summary 448 

Bibliography 451 



419 




Pig. 95, DISEASED CABBAGE PLANT SHOWING THE THIN STALK AND THE ABSENCE 

OF A HEAD 



STUDIES ON CLUBROOT OF CRUCIFEROUS PLANTS^ 

Charles Chupp 

Such an extensive literature on clubroot of cruciferous plants has accu- 
mulated that it would seem impossible for any one point to have escaped 
careful consideration. - But when a close examination* is made of all the 
data, it soon becomes apparent that only such prominent phases as 
symptoms, cytology of the organism, and control methods, have been 
dealt with extensively, while certain other less conspicuous features have 
been neglected. There still remain to be satisfactorily solved the follow- 
ing problems: (a) the part played by swarm-spores in the dissemination 
of Plasmodiophora Brassicae Wor., the organism that causes clubroot; 
(b) spore germination; (c) the manner in which the pathogene enters the 
host; (d) the distribution of the organism thruout the tissues of the root; 
(e) formation and size of the spores; and (f) the relation of bacteria to 
the normal development of the myxomycete. It is for the solution of 
these problems that the following investigations have been conducted. 

DISSEMINATION 

In a general way the manner in which the spores are carried is known, 
altho two errors are often met with in popular descriptions. For example, 
in a number of reports (Atkinson, 1889, Carruthers, 1893, ^^^ others) ^ 
are statements implying that swarm-spores swim about in the water 
of the soil until they reach a cabbage root. In a way this is correct, 
but the average layman at once pictures the swarm-spores as traveling 
from row to row of plants or even from field to field. Nothing could 
be more erroneous, for, as far as dissemination is concerned, the motility 
of the swarm-spore plays such a slight part that it need not be considered. 
Its energy is not directed in a straight line, and the very minuteness of the 
organism woidd preclude any effective locomotion in the time that it 
remains alive. 

In order to test the distance to which swarm-spores may travel in the 
soil, a box two feet square was filled with clay mixed with muck soil, 
and diseased roots were buried in one end. Cabbage seeds were then sown 
in the box, care being taken not to transfer any of the soil from the place 
where the inoculum was inserted. When the seedlings over the area 



' Also presented to the Faculty of the Graduate School of Cornell University, September, 1916, as a 
major thesis in partial fulfillment of the requirements for the degree of doctor of philosophy. 

Acknowledgment. The author gratefully acknowledges the helpful suggestions and criticisms offered 
him by Professor H. H. Whetzel and others in the Department of Plant Pathology at Cornell University. 

- Dates in parenthesis refer to bibliography, page 45 1. 

421 



422 



Bulletin 387 




Fig. 96. DISEASED CABBAGE SEEDLINGS 



Studies on Clubroot of Cruciferous Plants 423 

where diseased roots were buried had become so badly infected that they 
began to wilt and turn yellow, all the plants were discarded and the plat 
was reseeded. Different crops of seedlings were thus grown for almost 
a year, and, altho there was a gradual spread of the organism, it was 
only by careless watering and planting that the pathogene was carried 
in the soil to all parts of the box. 

Cabbage seeds were sown in a greenhouse plat in rows ten inches apart, 
the bottom of each trench being first lined with infested soil. Halfway 
between these rows were sown other rows, in the, trenches of which no in- 
fested soil was placed. The inoculated plants (fig. 96) became infected 
at a very early stage, while the plants that were only five inches away 
from the spores remained healthy until they were almost mature. 

A few authors (Carruthers, 1893, and others) claim that wind is an 
important agent in spore dissemination. This may be true in light, 
loose soil, and in localities where strong winds prevail, but in none of the 
observations made by the writer was there a single case in which the 
presence of the organism could be explained on this basis. On the other 
hand, many of the fields showed that if the soil were not transferred 
by some agent other than the wind the pathogene did not spread. On 
Long Island, New York, a certain field was observed, one corner of which 
was slightly lower than the adjoining part. This corner had been used 
for a garden until clubroot became so prevalent that the plat was no longer 
profitable for the raising of crucifers. It was then tilled with the remainder 
of the field for three years while various crops were grown, cabbages 
not being planted again until the fourth year. A space only slightly 
larger than the original garden then displayed the presence of clubroot. 
If wind had been an important agent it would have had an opportunity 
here, for the land was almost level and the soil was very loose. This 
was only one of several, cases in which the same conditions were observed. 

SPORE GERMINATION 

Very few persons have been successful in germinating the spores of 
Plasmodiophora Brassicae, and of those few who have been so fortunate, 
still fewer have seen the actual process. Woronin (1878) gives a brief 
description and a series of illustrations which have been copied by nearly 
all later writers on this phase of the subject. The general experience, 
however, seems to have been like that of Maire and Tison (19 11) while 
working with Tetramyxa parasitica Goebel. They saw only one spore 
actually germinating, and after a very long, tiresome vigil they left it 
for a few minutes. On returning from their temporary absence they found 
that the phenomenon had been completed. Notwithstanding these diffi- 



424 Bulletin 387 

culties, Eycleshymer (1894) not only found swarm-spores, but also found 
that when left in the culture for a few days these apparently fused 
into larger bodies, thereby reacting in much the same manner as Kunkel 
(191 5) found to be the case with Spongospora subterranea (Wollr.) 
Johnson. Kunkel discovered that each cell of a spore ball produces a 
single uninucleate amoeba which soon fuses with others of its kind to 
form a small plasmodium. This occurs not only in the case of spores in 
the soil, but even with those still in the base of the old sorus. 

There are several obstacles to be encountered in trying to observe the 
actual emergence of the protoplasmic mass from the old spore wall. First, 
it is difficult to get a very large percentage of germination unless the most 
favorable conditions are present. Secondly, all observations must be made 
with the oil-immersion objective. When the protoplasm is about half- 
way out, the spore wall and the emerging protoplast begin to move, making 
it hard to keep them in focus or even within the field. Consequently, 
when the process seems almost complete there is a sudden swift whirl, 
and the swarm-spore, with the adhering empty wall, darts out of sight. 
When located again, the spore wall is empty, and the swarm-spore, lost 
among others, is impossible of identification. For this reason no actual 
separation of the protoplasm from the spore wall has been seen, but 
enough of the process has been observed to enable investigators to deter- 
mine the general method by which this is accomplished and to be sure 
that a spore gives rise to only one swarm-spore. 

It was soon learned that spores do not germinate well, if at aU, in dis- 
tilled water, and further that, altho from one to five per cent of the spores 
taken directly from a fresh root germinate in muck-soil filtrate, a much 
larger percentage of germination can be obtained by exposing the roots 
to freezing temperatures for two weeks or longer. This was accomplished 
by tying the roots in cheesecloth and burying them under the snow, or 
in summer by keeping them in the refrigerator for that length of time. 
Drying the roots also seems to have a beneficial effect on germination, 
altho this must not be carried to the extreme. The muck-soil filtrate 
was made by filling an ordinary flowerpot with muck, placing it over a 
large funnel lined with filter paper, and then pouring hot water on the 
soil. The resulting medium was of an amber color and slightly acid. 

Temperature conditions also influence germination of the spores. It 
was practically impossible to obtain infection in the greenhouse during 
the coldest winter months when the temperature was from 10° to 18° C. 
The spores also fail to germinate at ordinary room temperature (from 1 6° 
to 21° C). The optimum temperature for germination proved to be from 
27° to 30° C. This, however, is not the case when spores are placed in 
test ttibes on agar with young cabbage seedlings, for under such conditions 



Studies on Clubroot of Cruciferous Plants 425 

infection takes place at a temperature of from 16° to 21° C. The pres- 
ence of the host seems in some manner to exert an influence which to a 
certain extent takes the place of that offered by a greater amount of heat. 

Usually the first sign of germination is a swelling of the spore, which 
sometimes becomes a third larger. This occurs within a period of from 
fifteen minutes to eight hours after the spores are placed in the medium, 
altho the best time for examining the culture proved to be at the end of 
six hours. After the swelling of the spore there is a bulging at one side. 
The protoplasm withdraws from near the opposite wall and leaves a 
nearly hyaline semicircle about two-thirds of the distance from the center. 
The pressure exerted splits the wall just enough to permit the protoplasm 
to ooze out. Unlike Woronin (1878) and Mangin (1902), the writer has 
never observed the protoplasm taking the various shapes that these 
authors assign to it, but while oozing out it collects in a sphere or a hemi- 
sphere against the wall on the outside. When about half of the proto- 
plasm has escaped, the whole body becomes motile. At first there is only 
a trembling, which gradually increases in violence until the spore is turned 
around entirely. The activity now becomes so great that it is with diffi- 
culty that the microscope is kept focused on it correctly. The final struggle 
is apparently a rapid spurt across the field, when the swarm-spore is lib- 
erated from its container and at once begins its rotatory activities. The 
whole process under the microscope consumes an hour or longer. Evi- 
dently the strong light turned on a spore retards the action, for in many 
cases the spores that had begun to germinate when placed in view showed 
no further signs of development, while those kept in the dark germinated 
much more rapidly and when examined at the end of the same period 
were found actively swimming about. 

A considerable part of the contents is left within the old spore wall, 
so that when the broken part is turned upward it has the appearance of 
a circle bounded by a darker band, the width of which is about one- third 
of the radius. If, however, the open part is on the side, the residue within 
the spore wall resembles more nearly a crescent (fig. 97). 

The swarm-spore when alive measures from r.7 to 3.5 m in length, 
being more or less pyriform with a thick flagellum at the smaller, or 
anterior, end and a vacuole near the posterior end. Unless stained, the 
flagellum cannot be seen under the microscope. The line of locomotion 
is never a straight one, for the flagelltmi is lashed about by the beak, 
which is constantly doubling backward so that a whirling motion is given 
to the swarm-spore. Altho the latter is a naked mass of protoplasm, 
the writer has never seen the various shapes which Woronin (1878, PI. 
xxxiv) has pictured; it was observed in every case to be globose or pyri- 
form, never having pseudopodia-like structures. 



426 



Bl'LLETIN 387 



It has been difficult to properly fix swarm-spores for staining flagella. 
The first method of staining tried was that ordinarily employed for bac- 
teria, namely, Loefflsr's mordant and Ziehl's carbol fuchsin. When 
bacteria were in the mount their flagella were stained, but those of the 

«P5^ swarm-spores had evi- 

m dently disappeared. The 

process was then modi- 
fied slightly, and the 
cover-glass mounts, in- 
stead of being left to 
dry in the incubator, 
were placed on slides in 
preparation dishes with 
ground-glass tops. In the 
bottom of each dish was 
placed a few cubic centi- 
meters of osmic acid, and 
the lid was then carefully 
fitted in place. The acid 
killed a few of the swarm- 
spores before the flagella 
could be withdrawn, but 
never a very large pro- 
portion. Besides demon- 
strating the presence of 
flagella, the stained ma- 
terial also displayed 
different stages of ger- 
mination (fig. 97). 

Kunkel ( 1 9 1 5) was able 
to get spore germination 
of Spongospora subter- 
ranea on an agar me- 
dium. Plasmodiophora 
Brassicae evidently does 
not react in the same 
way. During the three 
years of the present work, repeated efforts were made to secure not only 
germination on the surface of agar, but also formation of plasmodia. 
Unless the spores were immersed in water there was no development. 
They lay there until the agar became so dry that they finally lost their 
viability. If enough of the muck-soil filtrate was added, the swarm- 







Fig. 97. SPORES and swarm-spores of plasmodi- 
ophora BRASSICAE 

The two spores at the top have already germinated. The germi- 
nating spore and the two swarm-spores near the bottom were drawn 
from stained mounts. The bacillus shown is the form found oftenest 
in older diseased roots. X 2100 



Studies ox Clubroot of Cruciferous Plants 427 

spores appeared but there was no further development. They were 
active for a certain time, and then encysted and remained in that con- 
dition as long as the cultures were kept. This 
experiment was performed on four kinds of agar 
media, on potato plugs, and on healthy cabbage 
roots. In no case were there any signs of further 
growth. This, with subsequent infection experi- 
ments, indicates very strongly, if it does not prove 
positively, that the swarm-spores never fuse. 
This is in keeping with what has been found, or 
at least suggested, in all other cases of parasitic ^' 

slime molds, Spongospora suhterranea excepted. ] 

If spores for germination are taken from roots „ 

,, , , , . , . -,■ ■ r ■, -, Fig. 98. FLAGELLATE OR- 

that have not previously been dismrected, there ganisms a.ssociated 
are often found in the cultures flagellate bodies ^^'^th plasmodiophor.^ 

... , ,, , , , BRASSICAE 

which are almost small enough to resemble 

swarmspores. They are larger, however, are more active, and when stained 
are more or less reniform, having two fiagella arising from the concave 
side (fig. 98). These, as pointed out later, belong to another organism. 

PENETRATION 

In the knowledge of the life history of Plasniodiophora Brassicae, there 
has always been a gap between the swarm-spore stage and the amoeba 
within the cell, the true sequence of development never having been 
shown. Most writers pass over the difficulty with the mere statement that 
the organism enters the root and there begins its parasitic life. Woronin 
(1878), in this as in nearly all other points connected with clubroot, is 
the only one who has tried to fill in the gap. In a way he succeeded, but, 
as his plants died before reaching the stage in which invasion of any of 
the tissue took place, he is not sure that the root hair is the real point of 
entrance. He placed cabbage seedlings in shallow watch glasses, in water 
well supplied with spores. For some reason the plants began dying before 
hypertrophy took place. When the roots were examined microscopically, 
the root hairs were filled with amoebae but nothing further had happened. 
The question still remained, whether these infections under normal con- 
ditions would have been followed later by invasion of the cortical cells, 
or whether the case was similar to that which Schwartz (19 14) found in 
species of Ligniera. Schwartz thinks that penetration takes place near 
the apex of the root, so that when the root hairs act as bearers of the 
amoebae the parasite does not advance farther than the base of the cell. 

Most writers believe not only that the apical cells and the root hairs 
act as infection courts, but also that the epidermal cells can be infected 



428 Bulletin 387 

directly up to the time when the epidermal layer is thrown off (Woronin, 
1878). Somerville (1895) gives an observation as proof of this statement. 
He often found swellings high up on the roots of turnips, where he declares 
no root hairs could have been responsible for the entrance of the slime 
mold, which must have penetrated the thick cuticle. This question of 
entrance has a direct economic bearing on control, for, if Somerville's 
statement is true, Massee's (1903) assumption is certainly erroneous. 
Massee states that the Cruciferae can be attacked only during seedling 
time, and that after six weeks they are practically immune. It is doubtftd 
whether either Somerville or Massee interprets the conditions correctly. 
If infection could not take place after six weeks, the grower could control 
the disease merely by late transplanting and the proper care of his seed 
beds; but this has evidently not proved to be the case in practice. 

Maire and Tison (1909, 191 1) and vSchwartz (1910, 191 1, 1914) have 
done nearly all the work that has been reported on the parasitic slime 
molds other than Spongospora subterranea and Plasmodiophora Brassicae. 
It is interesting to note that their conclusions agree very closely, and that 
they feel sure the amoebae enter oftener thru the apical cells than otherwise, 
altho the root hairs also may serve as points of entrance. They made no 
particular study of this question, but were led to this conclusion by finding 
uninucleate amoebae in the cells near the growing tips. Their opinion is 
substantiated also by the presence of rows of diseased cortical cells, the 
divisions of which apparently take place when still very near the initial 
cells in the root tips. The powdery scab pathogene, Spongospora sub- 
terranea, passes directly thru and between the epidermal cells into the 
tuber (Kunkel, 19 15). 

There is more or less difhctdty in studying the nature of penetration 
in the case of Plasmodiophora Brassicae, because of the fact that the 
uninucleate amoebae are so small. They can be recognized only under a 
very high magnification, and, since they are so nearly transparent, stained 
sections must be used for all the work. A very large number of both 
longitudinal and cross sections were prepared, the thickness ranging from 
three to fifteen microns, and the staining was done with the combination 
stains of safranin, gentian violet, and orange G. These proved best for 
differentiating the parasite from the host, especially when orange G was 
used in excess. 

There is no possible stage in penetration that was not represented in 
the preparations. Large, as well as very small, roots were sectioned, 
and a great number of epidermal cells showed amoebae. But in a careful 
study of almost three hundred slides, none of these cells showed that 
penetration had taken place directly thru the cutinized wall. In a number 
of cases this appeared to be true when the sections were first examined, 



Studies on Clubroot of Cruciferous Plants 



429 



but a more detailed study of the same series showed the invaded cell to 
be in every case the basal portion of a root hair. This, together with the 
fact that no new swellings are ever found at any great distance from the 
region where root hairs might have existed previously, has led the writer 
to believe that seldom, if ever, is there direct penetration into simple 
epidermal cells. 

This holds true not only for the area above the place where the root 
hairs have disappeared, but evidently also for the space near the extreme 
'tips where the hairs have not yet been formed. Not only did these slides 
demonstrate this point, but infection secured under aseptic conditions 
in test tubes has confirmed it. The small root-tips were so placed that 
they were the first to come into contact with particles of diseased tissue 
and the muck-soil filtrate containing free spores. When these rootlets 
were sectioned and stained, they showed various stages of root-hair 
invasion, but no 
amoebae were 
found in any of 
the apical cells. 
The evidence pre- 
sented in these 
slides shows that 
these invasions 
are not, like those 
which Schwartz 
(19 1 4) suggested 
for Ligniera sp., 

confined alone to the epidermal cells of which the hairs are outgrowths. 
The passage of amoebse from the epidermal cells into the cortical tissue is 
demonstrated not only by the position of the amoebae within the paren- 
chyma cells, but also by actual cell- wall penetration. 

The argument advanced for other species of Plasmodiophoraceao, that 
infection must take place in the growing tip where cells are dividing 
rapidly because the organism often occurs in definite rows of the cortical 
cells, does not necessarily apply to Plasmodiophora Brassicae. A glance 
at a section of a root tip (fig. 99) indicates the difficulty that a swarm- 
spore woiold encounter in entering at this point. The rootcap does not 
merely protect the root tip, but a row of its cells extends upward almost 
halfway to the root hairs. The remaining distance is protected by a 
comparatively heavy cuticle, leaving the root hair as practically the only 
vulnerable point. Moreover, the presence of the organism in continuous 
rows of cells can be explained in another manner. The condition shown 




Fig. 99. LONGITUDINAL SECTION OF A CABBAGE ROOT 

This shows the tip of the cabbage root protected by the cells of the root- 
cap. X 1 10 



43° 



Bulletin 387 



in figure 100, b, gives no indication as to where penetration occurred. 
Yet by moving the section the length of half a dozen cells, there is seen 
an uninterrupted connection of diseased tissue between this particular 

row and the epidermis 
(fig. 100, a). 

So far as the writer's 
observations go, there 
seems to be no question 
but that penetration 
does take place thru the 
root hairs, and thru 
these only. Eycle- 
shymer (1894) suggests 
that wounds caused by 
insects may provide a 
means of entrance for 
the parasite. This is 
altogether probable ; yet 
the writer has never 
observed any indica- 
tions of this condition, 
so that if it ever hap- 
pens it apparently does 
so very rarely. If cul- 
tures coiild be secured 
within pieces of healthy 
disinfected roots in test 
tubes, it would at least 
be evidence that such 
wound infection might 
take place. Pinoy(i9o5) 
removed small pieces of 
nealthy roots by means 
of sterilized pipettes, 
and by inoculating them 
with spores secured cul- 
tures of the organism. 




Fig. 100. 



DISEASED CORTICAL TISSUE OF A CABBAGE 
ROOT 

A, A row of diseased cortical cells; B, another row of diseased cor- 
tical cells connected with the epidermis by an unbroken hne of diseased orovidcd th C tubcS WCrC 
tissue. X no ^ 

sealed so that the aerobic 
bacteria were deprived of oxygen. His discussion of this point is some- 
what lacking in clearness. Besides, the time in which he claims spores 
were produced in the roots is unusually short. He gives it as five days, 



Studies on Clubroot of Cruciferous Plants 431 

which is the same time that under the most favorable circumstances it 
takes swarm-spores to pass thru the root hairs into the cortical tissue 
and to develop sufficient hypertrophy to be visible to the naked eye. 
Kleimenov (19 12) tried the same experiment and failed. In the writer's 
experiments it was also tried repeatedly, always with failure. If the cul- 
tures were kept free from bacteria the root underwent no change. If 
bacteria were added, the root became soft and foul-smelling, whether the 
test tubes were closed with cotton plugs or sealed with paraffin over 
cork or cotton stoppers. Sealing did not stop the growth of the bacteria, 
as Pinoy claims for his experiments. 

Altho authors poptdarly describe with some assurance various ways 
in which the organism may enter the host, no one has observed the real 
process. Even Woronin, who believed that the organism passes thru the 
root hair, was never able to demonstrate this clearly. Nevertheless he 
felt assured that it enters in the form of a uninucleate amoeba, and his 
opinion has been accepted by most investigators. A few workers, such as 
Worthington G. Smith (1884), maintain that the organism enters the 
root in the form of a Plasmodium, but this theory has never been accepted 
generally. The question was revived again when Kunkel (1915) studied 
the powdery scab of potato, in which the swarm-spores are found to fuse 
before attacking the host. 

There seems to be no doubt in the minds of Maire and Tison (191 1) 
and Schwartz (19 14) that all the other known parasitic myxomycetes 
enter immediately after the swarm-spore stage. This conclusion is based 
on the fact that many of the slides of these investigators show the uninu- 
cleate forms in the apical cells. There is no other theory that would explain 
this phenomenon, unless a single uninucleate amoeba of an infecting 
Plasmodium passes thru the intervening cell walls and spreads in this 
manner thru the tissue. This is improbable. 

Because of the diminutive size of the swarm-spore, the only satisfactory 
method for studying penetration appears to be by means of stained sec- 
tions of roots showing the earliest stages possible. In the first part of this 
work, young plants from the greenhouse were used, but none of the stages 
were young enough to give just what was desired. An attempt was then 
made to grow plants in large test tubes on screens so arranged that the 
roots were hanging in muck-soil filtrate containing a heavy suspension of 
spores. The roots did not develop well when immersed in the liquid 
medium, and but few root hairs were present. An attempt was then made 
to grow seedlings in soil, in flats six inches square, with diseased tissue so 
plentiful that none of the plants could escape infection. The roots were 
fixed and embedded at intervals before the time when ordinary symptoms 
became apparent to the naked eye. This gave nearly all the early stages 



432 



BVLLETIN 387 



of infection, but the adhering particles of soil, which eoiild not be washed 
off without sacrificing the hairs, not only were detrimental to the micro- 
tome knife, but also obstructed a clear view of the cell walls. Finally a 
method was devised whereby infected roots could be procured free from 
any other contamination. Diseased roots that contained spores but were 
not far enough advanced to be invaded by bacteria "were sterilized on the 
surface with mercuric chloride and transferred to agar slants in test tubes. 
After two weeks cooling in the ice chest they were finely minced in the agar, 

and incubated 
until it was clear 
that no bacteria 
were present in 
the tissue, from 
which they might 
have been liber- 
ated by the cut- 
ting. After enough 
time had elapsed 
to insure perfect 
freedom from any 
saprophytes, a few 
drops of sterilized 
muck-soil filtrate, 
and a young cab- 
bage seedling 
which had been 
grown from disin- 
fected seed on agar 
in a petri dish, 
were added. It 
was necessary to 
exercise care in 
adding sufficient 
liquid to permit spore germination and not have an excess, which would 
injure the root. A few drops would not evaporate until all the swarm- 
spores had ample time to be set free and attack the root hair. The 
process was somewhat long, and very often roots were chosen which were 
too old and were already contaminated with bacteria. In spite of all the 
difficulties, enough pure cultures were obtained to provide a large number 
of sections which showed all sizes of amoebse. 

The first and most important thing shown by the stained sections was 
that Plasmodiophora Brassicae enters the root hair as a uninucleate amoeba, 





Fig. 1 01. THE AMCEBA OF PLASMODIOPHORA BRASSICAE IN A 
ROOT HAIR 

A, A root hair with an amcEba showing two nuclei. B, A uninucleate 
amoeba in a root hair which shows an abnormal swelling in the immediate 
vicinity of the organism. C, A uninucleate amoeba in a tangential section 
of a root hair; the nucleolus has elongated, as it ordinarily does just before 
nuclear division. D, A host nucleus in a root hair, showing its size as com- 
pared with that of a uninucleate amoeba. E, A uninucleate amoeba in a 
shrunken, distorted root hair. X 1600 



Studies on Clubroot of Cruciferous Plants 433 

not as a Plasmodium. There are several facts that prove this conclu- 
sively, even tho the actual phase of the organism passing thru the wall 
was never observed with certainty. A number of slides show cases that 
might be interpreted as actual penetration, but as the nucleus in 
no case appears in the act of making the passage one cannot be 
certain of such an interpretation. Nevertheless, numerous cases are 
to be found of a uninucleate amoeba just within the wall of the root 
hair and far enough away from any other infection to preclude all 
possibility of its having reached there except by entering singly thru the 
wall (fig. loi). 

Evidently the reason why no one has recorded this stage heretofore is 
because the amoeba hardly enters before nuclear division and growth 
takes place. Some slides show binucleate amoebae still within the hollow 
of the enlarged cavity, apparently produced by the stimulus of the para- 
site. Other sections show trinucleate amoebae, and it is not difficult to 
find amoebas with six or more nuclei (fig. 104, page 436). 

This series of stages would indicate that penetration takes place in 
the uninucleate stage, particularly since the large multinucleate amoebas 
are to be found, in nearly every instance, near the base of the root hair, 
while the smaller and fewer-nucleate amoebae are always on the inside of 
the root-hair wall about two-thirds of the distance from the base. Amoebae 
are seldom found in the tip of the hair. 

Another point that confirms the above view of penetration is that in 
the absence of growing host roots the swarm-spores develop no further 
when the spores are germinated under artificial conditions, and after a 
short period of activity the swarm-spores encyst and eventually die. 
If Plasmodia are formed under normal conditions, there should have been 
at least a suggestion of this in a few of the numerous cultures used in 
the experiments. 

In this connection also the very interesting question of sexual fusion 
arises. It is believed by several cytologists that there are two nuclear 
divisions just before spore formation and that one of these is probably a 
reduction division. If this is true, it would imply that somewhere in the 
life cycle there has been a fusion. Winge. (1913) and others believe that 
this occurs among the swarm-spores before they enter the host. Prowazek 
(1905) is of the opinion that the amoebae within the host unite and then 
the nuclei fuse. Even Nawaschin (1899) believes this union takes place, 
but apparently he thinks it is of no significance in reproduction. Maire 
and Tison (1909, 191 1) have disproved the amoebal union, and their view 
is certainly correct, for it is possible to find slides showing one amoeba 
breaking up into spores while in another, immediately adjoining, di\nsion 



434 



Bulletin 387 



has not yet begun (fig. 102, d). On the other hand, it would seem that the 
fusion of two swarm-spores would give an increase in size, but the measure- 
ments of amoeba just after penetration show them to be no larger than 
the swarm-spores just out of the spore wall. Consequently Winge's theory 




Pig. 102. SPORES and amceb^e of plasmodiophora brassicae 

A, Spores before their final separation from one another; B, cell filled with amoebae; C, cell filled with 
spores^ All X 800. D, Formation of spores, X Soo 

must be discarded. It thus appears that the real fusion stage, if there is 
one, is still to be discovered. 



distribution within the host tissues 

As stated above, the uninucleate amoeba, just after its entrance into the 
host, lies at first in a small cavity produced by the outward swelling of 
the part of the root hair at the point where the organism entered. This 
protuberance is no doubt caused by the irritating presence of the para- 
site (fig. 10 1, A, B, e). Following penetration the amoeba increases in size 
and pushes toward the center of the hair. The movement is accomplished 
by an actual amoeboid creeping, and an elongation and gradual segmen- 
tation of the forward ])art. W.oronin (1878) was able to observe the 



Studies on Clubroot of Cruciferous Plants 



435 



former method of locomotion in the living cells, and mentions it as the 
means by which the organism moves. Schwartz (19 lo), on the other 
hand, observed the growing of the amoeboid tip in Ligniera Junci (Sch.) 
M. et T., and explains the change of position on that basis alone. A root 
hair is shown in figure 104, d, which apparently was infected near the tip, 
and as the organism grew rootward fission took place, so that when the 
anterior part of the amoeba eventually reached the base of the cell the 
root hair was filled completely with the meronts, as Maire and Tison 
(191 1) designate the segmented parts (fig. 103). This does not always take 
place, for there were many more cases observed in which the intact 
amoeba reached the base of the cell (fig. 104, e, f). In either case, if the 
time consumed is too long, or if | — - — ^^^ 
for any other reason sponilation L^ ' j 
begins, the amoeba loses its 
power of further penetration 
into the cortical tissues. If, 
however, it reaches the inner 
wall of the root-hair cell, its 
pseudopodia are extended into 
the very smallest thread-like 
])rocesses, which pass thru and 
into the cortical cell (fig. 105, 
E, f,g). Schwartz (19 10), in de- 
scribing penetration by Ligniera 
Junci, gives the same route of 
invasion but does not state how 
the passage from the epidermis 
into the cortical cells takes 
place. This question is of es- 
pecial interest, since in the 
latter part of his discussion Schwartz states his belief that amoebae never 
have the power of penetrating cell walls. There is no other apparent 
means by which this could be accomplished, for the epidermal cells seldom 
divide periclinally. 

It would be difficult to explain the wide distribution of the parasite 
within the root if cell-wall penetration did not occur, even tho it were 
taken for granted that invasion begins in the apical cells. The rootcap 
S3 fully protects these rapidly dividing primary cells that one must pre- 
suppose that in order to reach them the organism can pierce the walls. 
Then, in the maturer roots constant secondary thickening by the cam- 
bium takes place, which would ultimately push most of the diseased cells 
toward the periphery or isolate them near the center. This, however, does 




Fig. 103. 



PHOTOMICROGRAPH OF CELLS CONTAIN- 
ING AMCEB^ 

One amoeba has elongated considerably and is separating 
into meronts 



436 



Bulletin 387 



'- ,<A^ ' 












Pig. 104. SECTIONS of cabbage root hairs showing amceb^ 

a .='?;;.l^''.,^SS!'t '!iT''^'''^ ^^ ^•^^'°?. '" ^/°°^ ¥^'"- .S- ^ '""'^^ distorted and swollen root hair, mth 
a small amoeba partly surrounding its nucleus, which is also much enlarged. C, An amoeba near the tip 
fi le/with ri'Jr^^t "'"^p"*' are elongated, as they ordinarily are just before nuclear division. D, A root ha^ 
PhnntT. H L '■• ^ ^"^"^ ^' ^"^«ba in epidermal cells of the root ^nd at the base of root hkirs; amoebi 
about to break up into spores. G, A root hair filled with an amoeba. H, A root hair filled with amnebS 
breatang up into spores: the vacuolar channels between each nucleus arc plainly vis^^blex 600 



Studies on Clubroot of Cruciferous Plants 



43 7 




Fig. 105. AMCEB^ IN THE HOST CELLS 

A, B, C, D, and G, Amoebae, with pseudopodia, in recently infected roots. E, Amcebas in adjoining 
cells, divided only by the cell walls; this is evidently a case in which penetration occurred, altho no con- 
necting strands are visible. F, Amoeba penetrating the cell wall. X HO 



438 



Bulletin 387 




Fig. 106. FORMATION OF "krank- 

heitsherde" 

All the narrow elongated cells are still 
un invaded, but they have increased greatly 
in number thru outward pressure of the 
hypertrophied cells. X no 



not happen, as may be seen by examination 
of cross-sections. Besides, if it did, it 
would explain only the presence of longi- 
tudinal rows of diseased cells, and not nec- 
essarily the whole " Krankheitsherde." For 
example, in figure 106 the original cortex 
was five cells wide. That is the same nimiber 
as is found in the " Krankheitsherde." 
These are connected by a single row of 
diseased cells. How could the diseased 
area have originated without direct migra- 
tion and still show no radial hyperplasia? 

Woronin's (1878) view is that the para- 
site, taking advantage of the pits found 
in the parenchyma, goes directly from cell 
to cell and thus thruout the root, much 
like Spongospora subterranea in tubers as 
described by Kunkel (191 5) except that the 
organism in the potato is intercellular. To 
Nawaschin (1899), who saw no actual pas- 
sage thru the walls, it seemed too difficult 
a task for the amoeba to break thru the 
plasma membrane; hence he decided that 
there is never any migration, the distri- 
bution being due entirely to rapid division 
of diseased cells. 

Maire and Tison (1909, 191 1) and 
Schwartz (1910, 1911, 1914), who have 
made observations on the other Plasmo- 
diophoraceae, explain the scattered diseased 
areas as due to infection of the apical cells 
which by subsequent divisions gives rise to 
the diseased rows so often seen. Schwartz 
(191 1), in spite of the fact that he saw 
pseudopodia in Sorosphaera graminis ex- 
tending thru the cell wall, makes the state- 
ment that he does not believe species of any 
of the genera show direct migration. He 
explains his skepticism on the ground 
that he never saw any accompanying 
nucleus in these pseudopodia. 



Studies on Clubroot of Cruciferous Plants 



439 



Lutman (19 13) figures actual passage thru the wall. He believes that 
the amoebge are transferred in the cortical tissue both by penetration and 
by division of the host cells. 

It is altogether possible to cut a large number of sections without obtain- 
ing any definite clue as to the mode of migration from the root hair to the 
cortex or the medullary ray, for in the later stages the cell wall acts as a 
perfect barrier. In view of this fact, Nawaschin might have done enough 
staining to complete his carefully planned cjrtological problem without 
once cutting a root so recently infected that the passage from one cell to 
another could be detected. During the first two years of the writer's 
study, only roots that showed evident hypertrophy were used and none 
of these gave any evidence of such a passage. As soon as the smallest 
rootlets were sectioned longi- 
tudinally, penetration could be 
observed. It is true that it never 
appeared abundantly; yet it 
might have been there and not 
noticed, for the opening in the 
wall is so minute and the strand 
which passes thru is so nearly 
hyaline that only deep staining 
will make it apparent under the 
microscope (fig. 105, f, page 437). 
There are numerous cases in 
which it is probable that such a 
migration has taken place but 
the connecting strand cannot be 
seen (fig. 105, e). 

The objection has been sug- 
gested that these strands are merely the remains of a thread which 
was not severed when the wall was laid down between two dividing cells. 
This may be true in such cases as are represented in figure 107, but in 
other cases the position of the cells precludes the tenability of such an 
assumption. 

Another argument in favor of cell-wall penetration is the shape of the 
amoeba in the initial stages of invasion as compared with that in later 
stages. When the smallest rootlets, containing only a few diseased cells, 
are sectioned longitudinally, the amoebae are usually seen to be elongated 
and often have pseudopodia extending in different directions. This is 
never true in a more advanced stage. The amoebee are then nearly 
spherical and remain stationary in the cell. This difference is seen on 
comparison of figures 105 and 108. 




Fig. 107. AMCEBA EXTENDING FROM ONE CELL 
INTO ANOTHER 



440 



Bulletin 387 



The small offspring at once begins to grow in the newly invaded cell, 
the process of penetration being repeated while the tissue is still young. 
From this statement, however, it is not to be inferred that all this occurs 
while there is no cell division, and that each daughter cell in turn does 
not become infected. Cell division certainly does take place from the 
beginning, first in conjunction with penetration and later alone. The 
result of both methods of invasion is illustrated by figure 100, b (page 430), 
which shows a row of eight diseased cells. They extend the same length as 
three healthy cells. Their relative lengths had been attained before 
infection occurred; therefore the organism must have passed thru at 
least two walls, while cell division accounts for the remainder. 

This leads the study to the 
process of " Klrankheitsherde " 
formation. The whole subject 
has usually been dismissed with 
the arbitrary statement that a 
single cell becomes diseased and 
then a closely packed group of 
cells finally results by repeated 
divisions both anticlinally and 
periclinally. A cursory study of 
cross sections would naturally 
suggest such an explanation, for 
undoubtedly the diseased areas 
are arranged in more or less 
distinct groups. But again lon- 
gitudinal sections of young and 
recently infected rootlets may 
be used to clear up the diffictdty 
and show the initial stages of a typical " Krankheitsherde." 

The impression must be avoided that passage thru cell walls is so fre- 
quent that a single root-hair infection will suffice to spread the organism 
thruout the entire affected part of the root. There must be repeated 
infections, since the amoebae never migrate far, as the longitudinal sec- 
tions show. They may enter in almost a straight path as far as the endo- 
dermis. The invaded cells may then divide or merely increase in size. 
Meanwhile the adjoining healthy cells show abnormal division. Nawaschin 
(1899) explains this hyperplasia on the part of non-invaded cells as due 
to the mechanical outward pressure of the much-enlarged diseased cells. 
Eleven rows of uninvaded cells adjacent to a "Krankheitsherde" are 
shown in figure 106 (page 438); in the healthy part of the root there are 
only five rows of cells. 




Fig. 108. 



HOST CELLS FILLED WITH CLOSELY 
CROWDED AMCEB^ 



Studies on Clubroot of Cruciferous Plants 441 

For some unknown reason the amoeba in some cases may not penetrate 
as far as the endndermis, but, after having reached a certain row of the 
cortical cells, it may pass upward or downward in that row, cell division 
taking place as fast as invasion occurs. This produces such rows of cells 
as are illustrated in figure 100. The vertical direction may be changed at 
any time and at frequent intervals to a horizontal one, and the adjacent 
rows thus affected at once begin cell division in each direction. The result 
is a true " Krankheitsherde." Any one of these diseased rows, or several 
of them, may extend beyond another group of healthy cells, from which 
the organism again moves horizontally. This will cause a second " Krank- 
heitsherde," above or below the first and separated by the length of one 
or more healthy cells. This is illustrated in figure 106 better than it can 
be described. A single infection may in this manner give rise to from 
one to probably six "Krankheitsherde," with the intervening uninvaded 
cells much increased in number over those in the normal tissue. 

It has already been pointed out why this longitudinal movement is 
interpreted as the result of cell-wall penetration instead of as being due 
to mere division. The diseased area shown in figure 106 is only five rows 
of cells in width. The perfectly normal tissue of the same root shows 
exactly the same number of rows. There has been no periclinal division, 
and therefore direct migration must have taken place. 

All this occurs when the root is only a few millimeters in diaineter. 
Por some reason the walls finally become impenetrable, and the amoebag 
become more nearly globose and later are transformed into masses of 
spores. 

SPORE FORMATION AND SIZE 

For the purpose of this discussion the nuclear phenomena need not 
be included. The generally accepted explanation of a true mitotic division 
followed by vacuolar separation into individual uninucleate spores is well 
known. This separation is supposed to take place almost simultaneously, 
but stained sections do not always show this to be true. Stages from the 
amoeba to the mature spore are represented in figure 102, d (page 434). 
In this case there were repeated successive separations instead of simul- 
taneous fission, so that each amoeba is divided into two, then four, and so 
on until no two nuclei longer remain together. The unstained spores in 
figure 102, A, show the same method of formation. They may then be 
hexagonal or irregular in outline, and much larger than when mature, 
but they soon become spherical. 

It was surprising to note the wide difference between the actual size 
of the spores, and the measurements (1.6 yu) given by Woronin (1878) 
and nearly all succeeding authors. MoUiard (1909) gives the diameter 



442 



Bulletin 387 



of the spores as from 1.8 to 2.2 fi, and Pinoy (1907), altho he does not 
state directl3^ says in speaking of swarm-spores that they are from 3 to 4 m 
in diameter. 

The measurements made in connection with the present experiments 
agree more nearly with those of Pinoy for the swarm-spores. The spores 
in formation, when not yet spherical, measure from 2.5 to 6.9 /x in diameter, 
being much more variable than those that are older. The smallest mature 
spore measured was 1.9 /z, and the largest was 4.3 jx. These measurements 
include not only living spores but also those stained in various ways. 
The average was 3.3 m- 

A SIMILAR ORGANISM 

For some time the writer was at a loss for an explanation of the occasional 
presence of from tw-o to twelve strange nuclei in certain root hairs and 
epidermal cells (fig. 109, a). These are from 3 to 4 ^ in diameter. 




Fig. 109. AN UNKNOWN ORGANISM ASSOCIATED WITH PLASMODIOPHORA BRASSICAE 

A, Nuclear-like bodies in a root hair, probably swarm-spores of Olpidium Brassicae; B and C, an 
unknown organism in the epidermal cells of a cabbage root, probably Olpidium Brassicae. X 800 

being smaller than the nuclei of the host cells. The nucleoli have a 
much denser content than those of the host cells, and are much smaller 
and less prominent. They appear to be entire swarm-spores containing 
no visible cytoplasm; however, they do not resemble those of Plasmodi- 
ophora Brassicae, being larger, and, most important of all, not having the 
hyaline zone about the nucleolus which is so characteristic of the latter. 



vStudies on Clubroot of Cruciferous Plants 443 

Furthermore, amoeba-like bodies are found in the epidermal cells or the 
layer next to them, which also look very much like Plasmodiophora 
but seem to be inclosed in a delicate wall. Stages have been found in which 
each of these bodies has an appendage, or neck, which protrudes thru 
the outer epidermal cell wall (fig. log, b, c). The organism compares very 
closely with that described by Woronin (1878) as Synchytrimn Brassicae 
and later by Dangeard (1886) as Olpidium Brassicae. Positive proof of 
its identity is lacking. 

The chytrid never produces any hypertrophy or other outside symptoms 
by which a diseased plant can be recognized, so that specimens were 
found only accidentally. For this reason it was impossible to study 
the swarm-spore stage or the details of the life history of the organism. 

The organism evidently enters by way of the root hairs, and never 
penetrates far into the host. None of the invaded cells are changed in 
size or general appearance. Even the invaded root hair does not have 
that slight enlargement which has been mentioned in connection with 
the entrance of the uninucleate amoeba of Plasmodiophora Brassicae. 

The fungus can infect a plant that seems perfectly healthy. At least 
it is not a saprophytic form following the clubroot organism, as roots 
were sectioned which showed it alone. 

BACTERIA IN RELATION TO PLASMODIOPHORA BRASSICAE 

Pinoy's (1902, 1903, 1905, 1907) work with Myxomycetes in their 
relation to bacteria, and his subsequent suggestion that there is a true 
symbiosis between the two, represent a very interesting phase in the study 
of Plasmodiophora. It has long been held that certain saprophytic slime 
molds feed on accompanying organisms, and the data at hand seem entirely 
plausible. Lister (1894) has seen the ingestion of bacteria by active 
swarm-spores. The experiments of Vuillemin (1903), Nadson (1901), 
and Potts (1902) show that Dictyostelium mucoroides Bref. feeds directly 
on bacterial colonies and destroys a large number of these at fruiting 
time. 

The general conditions of subsistence governing saprophytic forms 
and those controlling parasitic organisms are not the same, however; 
so that from a priori reasoning it would seem justifiable to say that Plas- 
modiophora Brassicae needs no concomitant organism in its life cycle. 
Yet the case is not so clear, since, on examination of nearly every root 
that has been diseased for some time, such an organism is found to be 
present. When the surface of these roots is sterilized and placed in agar, 
they may show no indication of bacteria until they are cut in two and 
the fresh surface is placed in contact with the medium. Moreover, E. F. 



444 Bulletin 387 

Smith (1911) and Eycleshymer (1894), both careful workers, state that 
they saw these bacteria. This is also in accordance with what Maire and 
Tison (191 1 ) claim to be true for certain parasitic slime molds that are 
able to ingest unicellular algae present in their aquatic host; and with 
what Kunkel (1915) has demonstrated in the case of Spongospora sub- 
terranea grown on agar in which pure cultures of plasmodia become 
abnormal and die, while those with which bacteria are present live and 
thrive. 

All of Pinoy's (1905) experiments appear to corroborate his idea that 
a coccus form enters the root with the swarm-spore and lives in constant 
association with the parasite thruout its entire life cycle. He stained 
sections of the root and observed bacterial forms within the cells. They 
appeared so much like parts of the cell contents that the microscopical 
analysis had to be accompanied by cultural study. For this he procured 
diseased roots of Brassica sinensis measuring from eight to ten centi- 
meters in diameter, seared the outside, and cut plugs from the interior 
by means of a flamed pipette. When these plugs were planted in agar 
media, numerous colonies of bacteria soon appeared. To prove that 
these organisms were necessary for the development of the myxomycete, 
Pinoy placed spores of Plasmodiophora Brassicae in a large number of test 
tubes containing sterilized extract of roots. In two tubes the spores were 
accidentally not associated with bacteria and they failed to germinate, 
while in all the other tubes the spores did germinate. 

Pinoy's results are interesting, altho the work does not appear to be 
extensive enough to warrant the conclusion he has drawn. The following 
experiments were undertaken by the writer in further quest for facts bearing 
on this problem: 

Thruout three years of study almost five hundred petri-dish and test- 
tube cultures have been made from diseased roots of all sizes and ages, 
grown under various conditions and in widely separated localities. The 
ordinary method of procedure was to place the roots for ten minutes in 
mercuric chloride i-iooo; then, after rinsing them several times in sterilized 
distilled water, to break the roots open and remove bits of the diseased 
tissue from the broken surface by means of a flamed scalpel. The bits 
of roots were placed in a sterilized petri dish, where they were teased apart 
in a few drops of sterilized distilled water. Two successive dilutions 
were made, and these, together with the original drop in which the tissue 
had been crushed, were poured into nutrient agar media. 

All the results were uniform in that no bacterial colonies were obtained 
from the roots with young swellings. From the medium-sized swellings 
occasional colonies developed; and from the larger galls, especially when 
the epidermis had been broken, numerous colonies always appeared. 



Studies on Clubroot of Cruciferous Plants 445 

The fact that only the small swellings show no contamination might 
be attributed to the penetration of the mercuric chloride. In order to 
avoid this source of error, the time of treatment was reduced from ten 
minutes to five, and even to three, or was dispensed with entirely, the roots 
being soaked for three hours in water that had been standing over calcium 
hypochlorite for two hours and then decanted. The results were the same. 

Another possible hindrance to the appearance of colonies at first might 
have been the medium, which was nutrient agar. In order to eliminate 
this objection, later cultures were made both in potato agar and in a 
medium made from the extract of healthy cabbage roots like that used 
successfully by Kleimenov (191 2). No bacterial growths were obtained. 

Bacteria have been found in large roots similar to those that Pinoy 
used; but Pinoy obtained a coccus, while the most prevalent form in 
the cultures of the writer has been a very motile rod-shaped bacterium 
producing yellowish, opalescent colonies on the various media. In test 
tubes containing disinfected diseased roots this organism readily produces 
a soft rot and thus liberates the spores of the slime mold. It is well 
known that the epidermis is soon ruptured after swelling begins, and from 
all indications the conditions are propitious for the entrance of any 
organisms that may be in the soil. This is doubly true for any that 
find exposed cabbage tissue a favorable substratum on which to reproduce, 
as does evidently the bacterium mentioned above. These series of cultures 
tend to show that bacteria do not enter with the swarm-spore, as Pinoy 
(1905) believes, but that the disease must advance to a certain stage 
before the bacteria can gain entrance. The above experiments are 
perhaps in themselves not sufficient proof, especially since they bear 
on the negative side of the question. To these, however, are to be added 
the following data: 

The writer has found that spores germinate better if they have been 
exposed to cold or to drying for a short time before being placed in a 
warm oven at a temperature of from 2 7° to 30° C. ; and that the best medium 
tested is water that has been filtered thru muck soil. Accordingly diseased 
roots were washed, treated with either mercuric chloride or calcium 
hypochlorite, placed in sterilized, cotton-plugged test tubes, and left 
in the ice box for seven days. At the end of that time they were cut 
into pieces ^vith a flamed scalpel and some of the sterilized muck filtrate 
was added, after which the roots were placed in the incubator for six 
hours. Before making mounts to examine the material, a loopfiil. of the 
filtrate was transferred to each of two petri dishes, which were then poured 
with nutrient agar. This was done in order to determine with certainty 
whether or not bacteria were present. Germination was fully as good 
when the bacteria were not present as when they were. This is in direct 



446 Bulletin 387 

opposition to Pinoy's (1905) statement that there is no development 
when the spores are not accompanied by a coccus. 

Diseased cabbage roots were disinfected with either mercuric chloride 
or calcium hypochlorite; if with the former, they were then rinsed three 
times carefully in different tubes of sterilized distilled water; if with 
the latter, they were rinsed in muck-soil filtrate, which is acid and tends 
to neutralize any of the calcium compounds that might adhere to the roots 
and retard germination of the spores. All the roots were then either 
transferred to tubes of nutrient agar slants or embedded in agar in petri 
dishes. If at the end of a week they showed no signs of contamination, 
those in the petri dishes were placed on agar slants, after which all the 
roots were minced and left for another week in order to make sure that 
no bacteria were in the roots and had been liberated by the mincing. 

Seeds of the cabbage were sterilized by the same method as was 
employed for the roots, but they were not rinsed in sterilized water when 
calcium hypochlorite was used. The seeds were planted in nutrient 
agar in petri dishes and the young plants were permitted to develop 
until they were free of the old seed coats. They were then placed in 
the tubes with the minced roots that showed no bacterial colonies, and 
a sufficient amount of the sterilized muck filtrate was added to insure 
spore germination but not enough to injure the small seedlings. 

This process, tho complicated and long, seems to fulfill all the require- 
ments that carefulness demands; and in the three series tried, from five 
to twenty per cent of the cultures were free from bacteria. The chief 
difficulty lies in the fact that there is such a narrow margin between spore 
formation and bacterial invasion that it is hard to select swellings which 
are neither too young to contain mature spores nor yet so old that bacteria 
have entered. • One objection to the experiment is obvious. There is 
no way of determining contaminations except by the absence of colonies 
on the agar where the roots have been minced and on which the seedlings 
grow. Yet it seems hardly possible that bacteria can be present thruout 
all these operations and not come into contact with the medium. Besides, 
where no bacterial colonies appear, the plants grow more vigorously, 
produce larger roots, and show infection sooner than in the contaminated 
tubes. Swellings apparent to the naked eye were formed at the end 
of five days the first time the experiment was tried. When the plants 
were fixed, sectioned, and stained, they showed amoebae in the cortex 
as well as in a large number of root hairs ; all of which tends to discount 
Pinoy's (1905) belief that there is no development of the parasites with- 
out a concomitant bacterium. 

Pinoy based his conclusions in part on the evidence presented by stained 
sections. Apparently he studied sections of large roots, since the roots 



Studies on Clubroot of Cruciferous Plants 



447 



that he received from Mangin were evidently eight or more centimeters 
in diameter. The writer was unable to procure thionine, the stain that 
Pinoy used, but he tried both Ziehl's carbol fuchsin and Kuehne's 
carbol methylene blue, which have always given good results in staining 
parasitic bacteria in other tissue. Parts of small, slightly swollen roots, 
as well as pieces of larger roots (of which some were still normal in color 
and others had begun to turn black), were fixed in Carnoy's fluid, con- 
sisting of glacial acetic acid and alcohol. The small, slightly swollen 
roots after staining showed no signs of bacteria. Pinoy (1905) states 







Fig. 1 10. s.\PROPHYTic organisms in diseased tissue 

A, Partly corroded starch grains between the amoebas, the refractive hila being the only 
visible part in some of them; B, bacteria in a cell of diseased tissue; C, mycelium of a sapro- 
phytic fungus in darkened diseased tissue. X 800 



that cocci appear as very refractive bodies among the amoebce. In this 
experiment, the hila of partly corroded starch grains (fig. no, a) appeared 
in several instances as spherical, brightly stained bodies; but they could 
hardly be mistaken for an organism, as the same effect is shown in healthy 
cells in which entire starch grains may be seen. 

The older, diseased tissue that has not yet turned dark presents a some- 
what different appearance from that of the yotingest swellings. The epi- 
dermal cells are torn in many places, and rod-shaped bacteria (fig. no, b) 
are found both within and between the cells. Many of these cells sho^^' 
broken passages in the walls where the organism could easily ha\'e entered. 



448 Bulletin 387 

In a blackened root the only additional change that can be recognized 
is the presence of hyphae. This blackness is almost a true criterion of 
the effects of a fungus, for the bacteria seldom, if ever, produce any 
pronounced discoloration (fig. no, c). 

It is not altogether a new phenomenon to find other organisms following 
parasitic slime molds. For example, the earlier writers who described 
Sorosphaera Veronica regarded it as a rust because of the mycelial threads 
which, according to these investigators, are constantly present. Maire 
and Tison (iQog) prove with but little difficulty that the fungi are merely 
saprophytic attendants. The case is almost identical with that which 
Schwartz (19 14) cites for species of Ligniera with which typical mycorrhiza 
are continual associates. 

It has been shown that non-parasitic myxomycetes undoubtedly make 
use of bacteria. It seems, therefore, altogether reasonable that when 
a facultative sa]jrophyte is grown under conditions to which Spongospora 
subterranea was subjected by Kunkel (191 5), it will assume the habits 
of a saprophyte. As far as this discussion is concerned, the only question 
is whether Spongospora subterranea still utilizes bacteria when in the 
potato tuber. 

Objection may be found to each of the above experiments taken alone. 
When considered together they cover the subject thoroly enough, and 
coincide so fully in their results that it seems logical to draw the con- 
clusion that Plasmodiophora Brassicae has no need for the bacteria and 
that the latter are merely attendant saprophytic forms which incidentally 
help to set free the spores of the parasite. Only two factors favor Pinoy's 
theory. One is the presence of bacteria in most roots in which any con- 
siderable swelling has taken place ; the other, the fact that there is a smaller 
number of different species of organisms present than might have been 
expected. ' Almost invariably the rod-shaped bacterium forming opalescent 
colonies on nutrient agar was the only one isolated. The facts, however, 
that spores can germinate in sterilized media, that infection can occur 
on seedlings in test tubes on nutrient agar where no bacterial colonies 
are present, and that recently infected roots never show bacteria either 
when tested in culture or under the microscope after staining, would 
seem to offset any evidence that heretofore has been adduced to the 
contrary. Therefore it seems evident that Plasmodiophora Brassicae is 
an obligate parasite, and, as such, needs no other food supply than that 
furnished by its host. 

SUMMARY 

Neither the motility of swarm-spores nor the action of winds is an 
important factor in the dissemination of Plasmodiophora Brassicae. 



Studies ox Clubroot of Cruciferous Plants 449 

Spores gemiinate better after a slight rest period and in such a medium 
as muck-soil filtrate. Each spore produces one swarm-spore, which, 
if not supplied with a host, develops no further. 

It is difficult to stain the flagella of swarm-spores, but if they are first 
killed instantly with fumes of osmic acid fairly good mounts can be 
obtained. 

Penetration takes place thru the wall of the root hair while the organism 
is in a uninucleate stage. The root hair at once shows hypertrophy. 
The amoeba increases in size as it passes rootward, and finally, by direct 
cell- wall penetration as well as by the division of the host cells, the patho- 
gene is distributed thruout the cortical tissue. 

The spores are not always formed by simultaneous vacuolar divisions 
of the amoebae, there being cases in which they are produced by successive 
divisions while the adjoining amoebae may still be in the nuclear stage. 

Aside from Plasmodiophora Brassicae, there is often present another 
organism, which causes no hypertrophy and which is probably Olpidium 
Brassicae (Wor.) Dang. 

In the experiments to determine the relation of bacteria to Plasmo- 
diophora Brassicae, a large number of isolations were attempted, diseased 
tissues of all stages were stained, spores were germinated in sterilized 
media, and infections were secured in test tubes under aseptic conditions. 
All this points to the fact that the bacteria do not enter the host as soon 
as the slime mold does, but follow only after there has been enough enlarge- 
ment of tissues to rupture the epidermis. Consequently the bacteria 
can be of no vital importance in the nutrition of the parasite. 



Studies on Clubroot of Cruciferous Plants 451 



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